Microorganisms of corynebacterium which can utilize xylose and method for producing l-lysine using same

ABSTRACT

The present invention relates to microorganisms of  corynebacterium  which can utilize xylose and to a method for producing L-lysine using same. More particularly, the present invention relates to microorganisms of  corynebacterium  which are modified, in which genes encoding xylose isomerase and xylulokinase which are xylose synthases are introduced to express the xylose synthase. The present invention also relates to a method for producing L-lysine, comprising a step of culturing the modified microorganisms of  corynebacterium  using xylose as a carbon source, and recovering L-lysine from the culture.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a Corynebacterium sp. microorganismmodified to utilize xylose and a method for producing L-lysine using thesame.

2. Description of the Related Art

Industrial microorganisms utilize sugar such as glucose, fructose, andsucrose as a carbon source. Agricultural products are usually used asfeedstock to obtain these carbon sources, but they are expensive and aremore valuable as food. Recently, instead of using agricultural productsas traditional feedstock, cellulosic biomass including agriculturalwaste or woody waste, industrial waste, etc. has attracted attention asan ideal sugar raw material for fermentation, because it has theadvantages of low cost and abundant supply.

Among them, xylose is the second most abundant lignocellulosiccarbohydrate in nature, and is a representative cellulosic biomass.Useful materials have been produced from xylose using industrialmicroorganisms. For example, a method of producing L-amino acid isknown, by culturing a Escherichia sp. strain in a medium containing amixture of pentoses including glucose and xylose, wherein the stain ismodified to increase expression of xylABFGHR gene cluster encoding anenzyme (xylosidase) hydrolyzing xyloside, which is a glycoside derivedfrom xylose, and then recovering L-amino acid from the medium (JapanesePatent No. 4665567).

On the other hand, a Coryneform bacteria, Corynebacterium glutamicum, isknown as a Gram-positive strain used in production of various L-aminoacids. As described above, because xylose is the second most abundantlignocellulosic carbohydrate in nature, it is expected that L-aminoacids such as L-lysine can be more economically produced fromCorynebacterium glutamicum by using xylose. However, Corynebacteriumglutamicum does not have important genes which are involved in themetabolic pathway of xylose, which is a pentose, and thus there is aproblem that L-amino acid cannot be produced from Corynebacteriumglutamicum by using xylose. To solve this problem, there has been areport that Corynebacterium glutamicum is modified to be able to utilizexylose by introducing xylose isomerase (XylA) and xylulokinase (XylB)derived from Escherichia coli (Kawaguchi et al., AEM 72:3418-3428,2006).

The present inventors have made extensive efforts to produce L-aminoacid in a more economical manner, and as a result, they found that whenXylA and XylB-encoding genes derived from Erwinia carotovora areintroduced into Corynebacterium glutamicum, the variant is able toutilize xylose to produce L-lysine and also shows more improvedxylose-utilizing ability than the previously known Coryneformmicroorganism introduced with xylA and xylB derived from Escherichiacoli, thereby completing the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a modifiedCorynebacterium sp. microorganism able to produce L-lysine by utilizingxylose.

Another object of the present invention is to provide a method forproducing L-lysine using the modified Corynebacterium sp. microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cleavage map of an expression vectorpECCG122-pcj7-xylAB(Er) of the present invention;

FIG. 2 shows a graph representing growth characteristics of a parentstrain and a transformant introduced with the expression vectoraccording to a carbon source contained in a medium; and

FIG. 3 shows a graph representing growth characteristics of a parentstrain and a transformant of which pcj7-xylAB(Er) is inserted onchromosome according to a carbon source contained in a medium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect, the present invention provides a modified Corynebacteriumsp. microorganism able to produce L-lysine by utilizing xylose,characterized in that activities of xylose isomerase and xylulokinasederived from Erwinia carotovora are introduced thereinto.

As used herein, the term “xylose isomerase (XylA)” means an enzymecatalyzing an isomerization reaction from xylose to xylulose, which isinvolved in the xylose metabolic pathway, and with respect to the objectof the present invention, it may be an enzyme derived from Erwiniacarotovora.

The XylA is xylose isomerase derived from Erwinia carotovora, and mayinclude a sequence capable of providing a xylose-utilizing ability byintroducing its activity together with activity of xylulokinase derivedfrom Erwinia carotovora into the Corynebacterium sp. microorganismhaving no xylose isomerase activity, without limitation. In addition, itis apparent that any sequence having an activity equivalent to that ofthe above sequence, although it is not derived from Erwinia carotovora,is included in the scope of the present invention.

For example, an amino acid sequence of SEQ ID NO: 1, or an amino acidsequence containing a conserved sequence of the amino acid sequence ofSEQ ID NO: 1 and substitution, deletion, insertion, addition orinversion of one amino acid or several amino acids (may vary dependingon positions and types of amino acid residues in the three-dimensionalstructure of the protein, specifically 2 to 20, specifically 2 to 10,more specifically 2 to 5 amino acids) at one or more positions, may beincluded. As long as it is able to maintain or enhance the XylAactivity, an amino acid sequence having 80% or more, specifically 90% ormore, more specifically 95% or more, much more specifically 97% or morehomology with the amino acid sequence of SEQ ID NO: 1 may be included,and the substitution, deletion, insertion, addition or inversion of theamino acid also includes a mutated sequence naturally occurring in themicroorganism having XylA activity or an artificially modified sequence.

As used herein, the term “homology” refers to identity between twodifferent amino acid sequences or two different nucleotide sequences,and can be determined by a method well known to those skilled in theart, for example, BLAST 2.0, which calculates parameters such as score,identity, and similarity, but is not particularly limited thereto.

As used herein, the term “xylulokinase” means an enzyme catalyzing aproduction reaction from xylulose to xylulose 5-phosphate, which isinvolved in the xylose metabolic pathway, and with respect to the objectof the present invention, it may be an enzyme derived from Erwiniacarotovora.

The XylB is xylulokinase derived from Erwinia carotovora, and mayinclude a sequence capable of providing a xylose-utilizing ability byintroducing its activity together with activity of xylose isomerasederived from Erwinia carotovora into the Corynebacterium sp.microorganism having no xylulokinase activity, without limitation. Inaddition, it is apparent that any sequence having an activity equivalentto that of the above sequence, although it is not derived from Erwiniacarotovora, is included in the scope of the present invention.

For example, an amino acid sequence of SEQ ID NO: 2, or an amino acidsequence containing a conserved sequence of the amino acid sequence ofSEQ ID NO: 2 and substitution, deletion, insertion, addition orinversion of one amino acid or several amino acids (may vary dependingon positions and types of amino acid residues in the three-dimensionalstructure of the protein, specifically 2 to 20, specifically 2 to 10,more specifically 2 to 5 amino acids) at one or more positions, may beincluded. As long as it is able to maintain or enhance the XylBactivity, an amino acid sequence having 80% or more, specifically 90% ormore, more specifically 95% or more, much more specifically 97% or morehomology with the amino acid sequence of SEQ ID NO: 2 may be included,and the substitution, deletion, insertion, addition or inversion of theamino acid also includes a mutated sequence naturally occurring in themicroorganism having XylB activity or an artificially modified sequence.

As used herein, the term “xylose isomerase (XylA)-encoding gene(hereinafter, xylA)” means a polynucleotide encoding the above describedXylA.

The gene may include a nucleotide sequence of SEQ ID NO: 3, a nucleotidesequence which can hybridize with a probe derived from the nucleotidesequence of SEQ ID NO: 3 under “stringent conditions”, or a nucleotidesequence, in which one nucleotide or several nucleotides is/aresubstituted, deleted, inserted, or added at one or more positions of thenucleotide sequence of SEQ ID NO: 3. As long as it is able to maintainor enhance the XylA activity, the gene may include a nucleotide sequencehaving 80% or more, specifically 90% or more, more specifically 95% ormore, much more specifically 97% or more homology with the nucleotidesequence of SEQ ID NO: 3, a nucleotide sequence substituted with codonsfavored by host cells, a nucleotide sequence of which N-terminus orC-terminus is extended or eliminated, or a nucleotide sequence of whichstart codon is modified to control expression level, and thus the geneis not particularly limited thereto.

As used herein, the term “xylulokinase (XylB)-encoding genethereinafter, xylB)” means a polynucleotide encoding the above describedXylB.

The gene may include a nucleotide sequence of SEQ ID NO: 4, a nucleotidesequence which can hybridize with a probe derived from the nucleotidesequence of SEQ ID NO: 4 under “stringent conditions”, or a nucleotidesequence, in which one nucleotide or several nucleotides is/aresubstituted, deleted, inserted, or added at one or more positions of thenucleotide sequence of SEQ ID NO: 4. As long as it is able to maintainor enhance the XylB activity, the gene may include a nucleotide sequencehaving 80% or more, specifically 90% or more, more specifically 95% ormore, much more specifically 97% or more homology with the nucleotidesequence of SEQ ID NO: 4, a nucleotide sequence substituted with codonsfavored by host cells, a nucleotide sequence of which N-terminus orC-terminus is extended or eliminated, or a nucleotide sequence of whichstart codon is modified to control expression level, and thus the geneis not particularly limited thereto.

As used herein, the term “stringent conditions” means conditions whichpermit a specific hybridization between polynucleotides, for example,hybridization in a hybridization buffer at 65° C. (3.5×SSC (0.15 MNaCl/0.15 M sodium citrate, pH 7.0). 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.5% SDS, 2 mM EDTA,2.5 mM NaH₂PO₄, pH 7), and a detailed description is disclosed in therelated art (Molecular Cloning (A Laboratory Manual, J. Sambrook et al.,Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold SpringHarbor, N.Y., 1989) or Current Protocols in Molecular Biology (F. M.Ausubel et al., Editors, John Wiley & Sons, Inc., New York).

As described above, introduction of XylA and XylB activities intoCorynebacterium sp. microorganism may be carried out by various methodswell known in the art. For example, there are a method of inserting apolynucleotide including the nucleotide sequences encoding XylA and XylBinto a chromosome, a method of introducing a vector system including thepolynucleotide into the microorganism, a method of introducing a potentpromoter to upstream of the nucleotide sequences encoding XylA and XylB,a method of introducing xylA and xylB with a modified promoter, or amethod of introducing a modified nucleotide sequences encoding XylA andXylB, or the like. More specifically, if the nucleotide sequencesencoding XylA and XylB are introduced, Corynebacteriumammoniagenes-derived pcj7 promoter (Korean Patent No. 10-0620092) can beused as a promoter for controlling the expression thereof. In oneembodiment of the present invention, acquisition of xylose-utilizingability was confirmed as the activity of the foreign gene absent in theparent strain was observed by introduction of an expression vector orchromosomal insertion.

As used herein, the term “vector” refers to a DNA product having anucleotide sequence of a polynucleotide encoding the target protein,which is operably linked to a suitable regulatory sequence to expressthe target protein in a suitable host. The regulatory sequence includesa promoter capable of initiating transcription, an arbitrary operatorsequence for regulating transcription, a sequence encoding anappropriate mRNA ribosome binding site, and sequences for regulating thetermination of transcription and translation. Once transformed into asuitable host, the vector may replicate or function independently of thehost genome, or may integrate into the genome itself.

The vector that is used in the present invention is not specificallylimited and may be any vector known in the art, as long as it canreplicate in a host. Example of the vector typically used may be naturalor recombinant plasmid, cosmid, virus and bacteriophage. For example, asthe phage vector or the cosmid vector, pWE15, M13, λMBL3, λMBL4, λIXII,λASHII, λAPII, λt10, λt11, Charon4A, Charon21A or the like may be used.As the plasmid vector, pBR type, pUC type, pBluescriptII type, pGEMtype, pTZ type, pCL type, pET type or the like may be used.

The vector useful in the present invention is not particularly limited,and the known expression vector may be used. Specifically, pACYC177,pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector or thelike may be used.

Furthermore, the vector used in the present invention may be a vectorcapable of transforming host cells, to insert the polynucleotideencoding the target protein into the chromosome of the host cell.Specific examples of the vector include, but are not limited to, theshuttle vector pECCG112 that can self-replicate in both directions in E.coli and or Coryne-type bacteria (Korean Patent No. 10-0057684).

As used herein, the term “transformation” means a series of operationsfor introducing a vector including a polynucleotide encoding a targetprotein into a host cell so as to express the protein encoded by thepolynucleotide in the host cell. The polynucleotide to be introducedinto the host cell may have any form, as long as it is introduced intothe host cell and expressed therein. For example, the polynucleotide maybe introduced into a host cell in the form of an expression cassettethat is a structure including all elements (a promoter operably linkedto the polynucleotide, a transcription termination signal, a ribosomebinding site, a translation termination signal, etc.) required forself-expression. The expression cassette may be in the form of aself-replicable expression vector. In addition, the polynucleotideitself may be introduced into a host cell to be operably linked to asequence required for expression in the host cell.

The host cell may be any one of prokaryotic microorganisms, as long asit is able to produce L-lysine. Examples of the host cell may includeProvidencia sp., Corynebacterium sp. and Brevibacterium sp.microorganism, specifically, Corynebacterium sp. microorganism, and morespecifically Corynebacterium glutamicum. In one embodiment of thepresent invention, when KCCM11016P, KCCM10770P, KFCC10750, and CJ3P asCorynebacterium sp. microorganism having no xylose-utilizing ability areintroduced with XlyA and XlyB derived from Erwinia carotovora, they areprovided with xylose-utilizing ability, and as a result, L-amino acidsuch as L-lysine can be produced by utilizing xylose as a carbon source(Tables 1 to 6).

Corynebacterium sp. microorganism having an ability to produce L-lysinemay be a variant resistant to an L-lysine analogue. The L-lysineanalogue inhibits growth of Coryneform microorganism, but thisinhibition is fully or partially desensitized when L-lysine coexists ina medium. Examples of the L-lysine analogue include, but are not limitedto, oxa-L-lysine, L-lysine hydroxamate,S-(2-aminoethyl)-L-cysteine(AEC), γ-methyl L-lysine, α-chlorocaprolactamor the like. Variant having resistance to these L-lysine analogues canbe obtained by a conventional artificial mutagenesis treatment toCoryneform microorganism. In addition, when genetic manipulation isconducted to induce L-lysine-producing microorganism, it can be achievedby improving the expression of one or more of genes encoding enzymesinvolved in the L-lysine biosynthetic system. Examples of these genesmay include dihydrodipicolinate synthase gene (dapA), aspartokinase gene(lysC), dihydrodipicolinate reductase gene (dapB), diaminopimelatedecarboxylase gene (lysA), diaminopimelate dehydrogenase gene (ddh),phosphoenolpyruvate carboxylase gene (ppc), aspartate semialdehydedehydrogenase gene (asd) and aspartase gene (aspA), but are not limitedthereto.

As used herein, the term “L-lysine” is one of the basic α-amino acids,is an essential amino acid that is not synthesized in the body, is oneof the L-amino acids, and has a chemical formula ofNH₂(CH₂)₄CH(NH₂)COOH. L-lysine is synthesized from oxaloacetate throughL-lysine biosynthetic pathway, and NADPH-dependent reductase catalyzesan intermediate process for L-lysine biosynthesis. During thebiosynthetic process of 1 molecule of L-lysine, 3 molecules of NADPH aredirectly consumed by the corresponding enzymes, and 1 molecule of NADPHis indirectly used.

As used herein, the term “Corynebacterium sp. microorganism capable ofproducing L-lysine” means Corynebacterium sp. microorganism modified toproduce L-lysine from xylose, which is prepared by introducing genesencoding the enzymes involved in the xylose metabolism and not found inthe Corynebacterium sp. microorganism. The Corynebacterium sp.microorganism may be, but is not particularly limited to.Corynebacterium glutamicum, and the enzymes involved in the xylosemetabolism may be, but are not particularly limited to, XylA and XylB.

In this regard, the host cell may be Corynebacterium sp. microorganism,in which expressions of one or more of the genes encoding enzymesinvolved in the L-lysine biosynthetic system are improved, and the genesencoding enzymes involved in the L-lysine biosynthetic system may be,but are not limited to, dihydrodipicolinate synthase gene (dapA),aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB),diaminopimelate decarboxylase gene (lysA), diaminopimelate dehydrogenasegene (ddh), phosphoenolpyruvate carboxylase gene (ppc), aspartatesemialdehyde dehydrogenase gene (asd), aspartase gene (aspA) or thelike.

In addition, the host cell may be a mutant strain resistant to anL-lysine analogue. The mutant strain may be obtained by mutation ofCorynebacterium sp. microorganism. The L-lysine analogue inhibits growthof Coryneform microorganism, but this inhibition is fully or partiallydesensitized when L-lysine coexists in a medium. Examples of theL-lysine analogue may be, but are not particularly limited to,preferably oxa-L-lysine, L-lysine hydroxamate,S-(2-aminoethyl)-L-cysteine (AEC), γ-methyl L-lysine,α-chlorocaprolactam or the like.

Meanwhile, in the present invention, activities of the known enzymesinvolved in the L-lysine biosynthesis may be additionally controlled inorder to further improve the L-lysine production. Specifically, in thepresent invention, asd, dapB, and ddh genes, each encoding aspartatesemialdehyde dehydrogenase, dihydrodipicolinate reductase anddiaminopimelate dehydrogenase enzymes, are overexpressed to additionallycontrol activities of the enzymes, thereby improving the L-lysineproduction.

According to one embodiment of the present invention, the presentinventors selected Erwinia carotovora (SCRI1043)-derived ECA0097(xylA)(SEQ ID NO: 1) and ECA0096(xylB) (SEQ ID NO: 2) as suitable genesencoding XylA and XylB to introduce into Corynebacterium sp.microorganism (Example 1), and they cloned the selected genes encodingXylA and XylB so as to construct an expression vectorpECCG122-pcj7-xylA-xylB (hereinafter, pECCG122-pcj7-xylAB(Er))expressing xylA and xylB (hereinafter, xylAB(Er)) at the same time. Theexpression vector was introduced into Corynebacterium glutamicumKCCM11016P (this microorganism was disclosed as KFCC10881, andre-deposited to an International Depositary Authority under the BudapestTreaty with Accession No. KCCM11016P. Korean Patent Nos. 10-0159812 and10-0397322) to prepare a transformant overexpressing xylAB(Er). It wasconfirmed that the prepared transformant grows by utilizing xylose as acarbon source (FIG. 2), and produces L-lysine by utilizing each ofglucose and xylose, or by utilizing glucose and xylose at the same time(Table 1). In addition, in order to express xylAB(Er) on the chromosome,a recombinant vector for chromosomal insertion, pDZTn-pcj7-xylAB(Er) wasconstructed, and transformed into KCCM11016P, and through secondcrossover, a transformant KCCM11016P-pcj7-xylAB(Er) having xylAB(Er)operably linked to pcj7 promoter inside the transposon on the chromosomewas constructed. It was also confirmed that the transformant grows byutilizing xylose as a carbon source (FIG. 3), and produces L-lysine byutilizing each of glucose and xylose, or by utilizing glucose and xyloseat the same time (Table 2). Furthermore, in order to compare the effectsof improving xylose-utilizing ability between introduction of thepreviously reported E. coli-derived xylAB (hereinafter, xylAB(Ec)) andintroduction of Erwinia carotovora-derived xylAB(Er) of the presentinvention, a strain (KCCM11016P-pcj7-xylAB(Ec)) was prepared byintroducing xylAB(Ec) into KCCM11016P, and its xylose-utilizing abilityand L-lysine production characteristics were compared with those of theprepared KCCM11016P-pcj7-xylAB(Er). As a result, it was found thatxylose consumption rate of KCCM1016P-pcj7-xylAB(Er) was remarkablyincreased, compared to that of KCCM11016P-pcj7-xylAB(Ec), indicatingimprovement in fermentative production of L-lysine (Table 3). Inaddition, in order to confirm whether various Corynebacterium sp.microorganisms show the same results, pDZTn-pcj7-xylAB(Er) wasintroduced to an L-lysine-producing strain KCCM10770P to prepare atransformant KCCM10770P-pcj7-xylAB(Er), and it was confirmed that thetransformant is able to produce L-lysine by utilizing each of glucoseand xylose, or by utilizing glucose and xylose at the same time (Table4). pDZTn-pcj7-xylAB(Er) was also introduced into anotherL-lysine-producing strain KFCC10750 (this microorganism was disclosed asKFCC10750, and re-deposited to an International Depositary Authorityunder the Budapest Treaty with Accession No. KCCM11347P, Korean PatentNo. 10-0073610) to prepare a transformant KFCC10750-pcj7-xylAB(Er). Itwas also confirmed that this transformant is able to produce L-lysine byutilizing each of glucose and xylose, or by utilizing glucose and xyloseat the same time (Table 5). Further, pDZTn-pcj7-xylAB(Er) was alsointroduced into the other L-lysine-producing strain CJ3P to prepare atransformant CJ3P-pcj7-xylAB(Er). It was also confirmed that thistransformant is able to produce L-lysine by utilizing each of glucoseand xylose, or by utilizing glucose and xylose at the same time (Table6).

Accordingly, the present inventors designated the transformant as“CA01-2195”, which grows by utilizing xylose in a medium and alsoproduces L-lysine by utilizing xylose and glucose in the medium, anddeposited it under the Budapest Treaty at the Korean Culture Center ofMicroorganisms (KCCM, located on Hongjae 1-Dong, Seodaemun-Gu, Seoul,Korea) on Dec. 29, 2011 with Accession No. KCCM11242P. That is, thisdeposit is recognized by an International Depositary Authority under theBudapest Treaty.

In another aspect, the present invention provides a method for producingL-lysine, including the steps of (i) culturing the modifiedCorynebacterium sp. microorganism able to produce L-lysine by utilizingxylose in a culture medium containing xylose as a carbon source so as toobtain a culture broth; and (ii) recovering L-lysine from the culturebroth.

As used herein, the term “culturing” means that a microorganism iscultured under artificially controlled environmental conditions. In thepresent invention, the method for culturing Corynebacterium sp.microorganism may be conducted using a method widely known in the art.Specifically, examples of the culturing method include batch process,fed batch or repeated fed batch process in a continuous manner, but arenot limited thereto.

The medium used for the culture has to meet the requirements of aspecific microorganism in a proper manner while controlling temperature,pH, etc. under aerobic conditions in a typical medium containing aproper carbon source, nitrogen source, amino acids, vitamins, etc. Theculture media for Corynebacterium strain are disclosed (e.g., Manual ofMethods for General Bacteriology, American Society for Bacteriology,Washington D.C., USA, 1981). Possible carbon sources may include sugarsand carbohydrates such as sucrose, lactose, fructose, maltose, starch,and cellulose, in addition to a mixture of glucose and xylose as a maincarbon source, oils and fats such as soy bean oil, sunflower oil, castoroil, and coconut fat, fatty acids such as palmitic acid, stearic acid,and linoleic acid, alcohols such as glycerol and ethanol, and organicacids such as acetic acid. These substances may be used individually oras mixtures. Possible nitrogen sources may include inorganic nitrogensources such as ammonia, ammonium sulfate, ammonium chloride, ammoniumacetate, ammonium phosphate, ammonium carbonate and ammonium nitrate;amino acids such as glutamic acid, methionine, and glutamine; andorganic nitrogen sources such as peptone, NZ-amine, meat extract, yeastextract, malt extract, corn steep liquor, casein hydrolysates, fish ordecomposition products thereof, and defatted soybean cake ordecomposition products thereof. These nitrogen sources may be usedindividually or in combination. The medium may include potassiumdihydrogen phosphate, dipotassium hydrogen phosphate or thecorresponding sodium-containing salts as phosphorus sources. Possiblephosphorus sources may include potassium dihydrogen phosphate,dipotassium hydrogen phosphate or the corresponding sodium-containingsalts. Further, inorganic compounds such as sodium chloride, calciumchloride, iron chloride, magnesium sulfate, iron sulfate, manganesesulfate and calcium carbonate may be used. In addition to the abovesubstances, essential growth substances, such as amino acids andvitamins, may be included.

Appropriate precursors may be also added to the culture media. Theabove-mentioned substances may be suitably added to the culture mediumin batch, fed-batch or continuous mode during cultivation, but are notparticularly limited thereto. pH of the culture may be adjusted bysuitably adding basic compounds such as sodium hydroxide, potassiumhydroxide, and ammonia, or acidic compounds such as phosphoric acid andsulfuric acid.

An anti-foaming agent such as fatty acid polyglycol esters may be usedto suppress the development of foam. In order to maintain aerobiccondition, oxygen or oxygen-containing gas (e.g., air) may be introducedinto the culture broth. The temperature of the culture broth is normally27° C. to 37° C., specifically 30° C. to 35° C. The cultivation may becontinued until the production of L-lysine reaches a desired level. Thisobjective may be normally achieved within 10 to 100 hours. L-lysine maybe released into the culture medium or included within the cells.

Furthermore, the step of recovering L-lysine from the culture broth maybe performed by a known method known in the art. Specifically, the knownmethod for recovering L-lysine is, but not particularly limited to,specifically centrifugation, filtration, extraction, spraying, drying,evaporation, precipitation, crystallization, electrophoresis,differential solubility (e.g., ammonium sulfate precipitation),chromatography (e.g., ion exchange, affinity, hydrophobic, and sizeexclusion) or the like.

Hereinafter, the constitutions and effects of the present invention willbe described in more detail with reference to Examples. However, theseExamples are for illustrative purposes only, and the scope of theinvention is not intended to be limited by these Examples.

Example 1 Selection of Foreign Gene

Erwinia carotovora (SCRI1043)-derived ECA0097(xylA) (amino acid: SEQ IDNO: 1, nucleotide: SEQ ID NO: 3) and ECA0096(xylB) (amino acid: SEQ IDNO: 2, nucleotide: SEQ ID NO: 4) were selected as foreign genes toprepare a modified Corynebacterium sp. microorganism provided with axylose-utilizing ability.

Example 2 Construction of Erwinia carotovora-Derived xylAB ExpressingVector

Erwinia carotovora-derived XylA and XylB-encoding genes selected inExample 1 are located close to each other. Information (Accession NO.BX950851) about xylA and xylB(Er) and surrounding nucleotide sequencewas obtained from US NIH GenBank, and based on the obtained nucleotidesequence, primers for amplification of Erwinia carotovora-derivedxylAB(Er) were synthesized.

SEQ ID NO: 5:  5′-ACACATATGCAAGCCTATTTTGAACAGATC-3′ SEQ ID NO: 6:5′-AGAACTAGTGCCTTTTGGTGGTGTTTAAGT-3′

In order to obtain the xylAB(Er) fragment, PCR was conducted usingchromosomal DNA of Erwinia carotovora strain SCRI1043 as a template anda pair of primers (SEQ ID NOs: 5 and 6). PfuUltra™ high-fidelity DNApolymerase (Stratagene) was used as the polymerase, and PCR wasconducted with denaturation at 94° C. for 5 minutes, followed byrepeating the cycle 30 times including denaturation at 94° C. for 30seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C.for 3 minutes, and then polymerization at 72° C. for 7 minutes. As aresult, a gene fragment of 3122 bp containing xylAB(Er) (SEQ ID NO: 17)of 2844 bp was obtained (SEQ ID NO: 18). In order to obtainCorynebacterium ammoniagenes-derived pcj7 promoter (KR0620092), PCR wasconducted using genomic DNA of Corynebacterium ammoniagenes CJHB100(KR0620092) as a template and a pair of primers (SEQ ID NOs: 15 and 16).PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as thepolymerase, and PCR was conducted with denaturation at 94° C. for 5minutes, followed by repeating the cycle 30 times including denaturationat 94° C. for 30 seconds, annealing at 56° C. for 30 seconds andpolymerization at 72° C. for 1 minute, and then polymerization at 72° C.for 7 minutes. As a result, a polynucleotide of 318 bp was obtained (SEQID NO: 14).

SEQ ID NO: 15: 5′-AATCTAGAAACATCCCAGCGCTA-3′ SEQ ID NO: 16:5′-AAACTAGTCATATGTGTTTCCTTTCGTTG-3′

pcj7 was cloned into E. coli-Corynebacterium shuttle vector pECCG122using restriction enzymes, XbaI and SpeI, and then xylAB(Er) fragmentwas cloned using NdeI and SpeI, thereby obtaining apECCG122-pcj7-xylAB(Er) vector (FIG. 1). FIG. 1 is a cleavage map of theexpression vector pECCG122-pcj7-xylAB(Er) of the present invention.

Example 3 Development of L-Lysine-Producing Strain Introduced withErwinia carotovora-Derived xylAB and Examination of Xylose-UtilizingAbility

Each expression vector pECCG122-pcj7-xylAB(Er) obtained in Example 2 wasintroduced into Corynebacterium glutamicum KCCM11016P (Korean PatentNos. 10-0159812 and 10-0397322) to prepare a xylAB(Er)-expressingtransformant, Corynebacterium glutamicum CA01-2195.

In order to compare the xylose-utilizing ability between KCCM11016P andCA01-2195, the strains were cultured in a seed medium containing glucoseor xylose as a carbon source and their growth characteristics werecompared. They were also cultured in a production medium containingglucose or xylose as a carbon source and their L-lysine productioncharacteristics were compared.

First, in order to compare the growth characteristics, the strains wereinoculated in 25 ml of seed medium [carbon source (glucose or xylose) 10g/l, peptone 10 g/l, yeast extract 10 g/l, urea 5 g/l, KH₂PO₄ 4 g/l,K₂HPO₄ 8 g/l, MgSO₄7H₂O 0.5 g/l, biotin 100 μg/l, thiamine-HCl 1 mg/l,pH 7.0], respectively. Absorbance (OD600) of the culture broth wasmeasured while culturing the strains at 30° C. for 32 hours, andcompared to each other (FIG. 2). FIG. 2 is a graph showing growthcharacteristics of KCCM11016P and CA01-2195 according to carbon sourcecontained in the medium, in which (♦) indicates KCCM11016P cultured inthe medium containing glucose, (▪) indicates KCCM11016P cultured in themedium containing xylose, (▴) indicates CA01-2195 cultured in the mediumcontaining glucose, and (x) indicates CA01-2195 cultured in the mediumcontaining xylose.

As shown in FIG. 2, there was no difference in growth characteristicsbetween KCCM11016P and CA01-2195 in the seed medium containing glucoseas a carbon source. However, in the seed medium containing xylose as acarbon source, CA01-2195 grew to a certain level whereas KCCM11016Phardly grew. Therefore, it can be seen that CA01-2195 is able to grow byutilizing xylose contained in the medium as a sole carbon source.

Next, to compare L-lysine production characteristics between KCCM11016Pand CA01-2195, 1 ml of seed culture broth was inoculated in 24 ml ofproduction medium [carbon source, (NH₄)₂SO₄40 g/l, soybean protein 2.5g/l, corn steep solids 5 g/l, urea 3 g/l, KH₂PO₄ 1 g/l, MgSO₄7H₂O 0.5g/l, biotin 100 μg/l, thiamine-HCl 1 mg/l, CaCO₃ 30 g/l, pH 7.0], andcultured at 35° C. and 200 rpm for 72 hours. At this time, glucose 100g/l, glucose 50 g/l+xylose 50 g/l, and glucose 70 g/l+xylose 30 g/l weredetermined to be used as the carbon source. Next, concentration ofL-lysine, concentration of residual xylose, and concentration ofresidual glucose in each culture broth were measured and compared (Table1).

TABLE 1 Glucose 50 g/l + Glucose 70 g/l + Glucose 100 g/l Xylose 50 g/lXylose 30 g/l Strain L-lysine R.X R.G L-lysine R.X R.G L-lysine R.X R.GKCCM11016P 42 — 0 21 50 0 29 30 0 CA01-2195 42 — 0 40  0 0 41  0 0 R.X:residual xylose (concentration of residual xylose after reactiontermination) (unit: g/l) R.G: residual glucose (concentration ofresidual glucose after reaction termination)

As shown in Table 1, when the medium containing no xylose (glucose 100g/l) was used, concentration of L-lysine produced in the parent strainwas equivalent to that of CA01-2195. However, when the medium containingxylose (glucose 50 g/l+xylose 50 g/l, and glucose 70 g/l+xylose 30 g/l)was used, CA01-2195 produced L-lysine by consuming both glucose andxylose whereas the parent strain produced L-lysine by consuming noxylose but glucose only.

This result indicates that Corynebacterium sp. microorganism having noxylose-utilizing ability is able to consume xylose by introducing withErwinia carotovora-derived xylAB.

Example 4 Construction of Recombinant Vector for Chromosomal Insertionof Erwinia carotovora-Derived xylAB (pDZTn-pcj7-xylAB(Er)) andRecombinant Vector for Chromosomal Insertion of E. coli-Derived xylAB(pDZTn-pcj7-xylAB(Ec))

To express the xylAB(Er) on chromosome, a recombinant vectorpDZTn-pcj7-xylAB(Er) for chromosomal insertion was constructed. Toobtain the pcj7-xylAB(Er) fragment, PCR was conducted usingpECCG122-pcj7-xylAB(Er) obtained in Example as a template and a pair ofprimers (SEQ ID NOs: 7 and 8). PfuUltra™ high-fidelity DNA polymerase(Stratagene) was used as the polymerase, and PCR was conducted withdenaturation at 94° C. for 5 minutes, followed by repeating the cycle 30times including denaturation at 94° C. for 30 seconds, annealing at 56°C. for 30 seconds and polymerization at 72° C. for 3 minutes, and thenpolymerization at 72° C. for 7 minutes. As a result, a gene fragment of3440 bp was obtained (SEQ ID NO: 9). Consequently, pcj7-xylAB(Er) of3440 bp was cloned into pDZTn vector (Korean Patent No. 10-1126041)treated with restriction enzyme SpeI using a BD In-Fusion kit, therebyobtaining a pDZTn-pcj7-xylAB(Er) recombinant vector.

SEQ ID NO: 7: 5′-GAGTTCCTCGAGACTAGTAGAAACATCCCAGCGCTA-3′ SEQ ID NO 8:5′-GATGTCGGGCCCACTAGGCCTTTTTGGTGGTGTTTA-3′

Next, to express E. coli-derived xylAB on chromosome, recombinantpDZTn-pcj7-xylAB(Ec) for chromosomal insertion was constructed. Toobtain pcj7 promoter, PCR was conducted using the pcj7 fragment obtainedin Example 2 as a template and a pair of primers (SEQ ID NOs: 7 and 10).PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as thepolymerase, and PCR was conducted with denaturation at 94° C. for 5minutes, followed by repeating the cycle 30 times including denaturationat 94° C. for 30 seconds, annealing at 56° C. for 30 seconds andpolymerization at 72° C. for 1 minute, and then polymerization at 72° C.for 7 minutes. As a result, a gene fragment of 318 bp was obtained. Toobtain xylAB(Ec) fragment, PCR was conducted using chromosomal DNA of E.coli K12 as a template and a pair of primers (SEQ ID NOs: 11 and 12).PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as thepolymerase, and PCR was conducted with denaturation at 94° C. for 5minutes, followed by repeating the cycle 30 times including denaturationat 94° C. for 30 seconds, annealing at 56° C. for 30 seconds andpolymerization at 72° C. for 3 minutes, and then polymerization at 72°C. for 7 minutes. As a result, a xylAB(Ec) fragment of 3145 bp wasobtained (SEQ ID NO: 13). Consequently, the PCR products of pcj7 regionof 318 bp and xylAB(Ec) of 3145 bp were cloned into pDZTn vector treatedwith restriction enzyme SpeI using a BD In-Fusion kit, thereby a finalpDZTn-pcj7-xylAB(Ec) recombinant vector.

SEQ ID NO: 10:  5′-TCAAAATAGGCTTGCATGAGTGTTTCCTTTCGTTG-3′ SEQ ID NO: 11:5′-CAACGAAAGGAAACACATGCAAGCCTATTTTGAC-3′ SEQ ID NO: 12:5′-GATGTCGGGCCCACTAGTGCTGTCATTAACACGCCA-3′

Example 5 Development of L-Lysine-Producing Strain Inserted withErwinia-Derived xylAB and Examination of Xylose-Utilizing Ability

The prepared pDZTn-pcj7-xylAB(Er) vector was transformed intoKCCM11016P, and through second crossover, a KCCM11016P-pcj7-xylAB(Er)strain having xylAB(Er) with substitution of one copy of pcj7 promoterinside the transposon on the chromosome was obtained.

To compare the xylose-utilizing ability betweenKCCM11016P-pcj7-xylAB(Er) and KCCM11016P, they were cultured in a seedmedium containing glucose or xylose as a carbon source and their growthcharacteristics were compared, and they were cultured in a productionmedium containing glucose or xylose as a carbon source and theirL-lysine production characteristics were compared, in the same manner asin Example 3.

FIG. 3 is a graph showing growth characteristics of the strainsaccording to carbon source contained in the medium, in which (♦)indicates KCCM11016P cultured in the medium containing glucose, (▪)indicates KCCM11016P cultured in the medium containing xylose, (▴)indicates KCCM11016P-pcj7-xylAB(Er) cultured in the medium containingglucose, and (x) indicates KCCM11016P-pcj7-xylAB(Er) cultured in themedium containing xylose.

There was no difference in growth characteristics betweenKCCM11016P-pcj7-xylAB(Er) and KCCM11016P in the seed medium containingglucose as a carbon source. However, in the seed medium containingxylose as a carbon source, KCCM11016P-pcj7-xylAB(Er) grew to a certainlevel whereas KCCM111016P hardly grew. Therefore, it can be seen thatwhen xylAB(Er) is inserted into the chromosome, the strain is able togrow by utilizing xylose contained in the medium.

Next, L-lysine production characteristics of KCCM11016P-pcj7-xylAB(Er)and KCCM11016P were examined and compared with each other (Table 2).

TABLE 2 Glucose 50 g/l + Glucose 70 g/l + Glucose 100 g/l Xylose 50 g/lXylose 30 g/l Strain L-lysine R.X R.G L-lysine R.X R.G L-lysine R.X R.GKCCM11016P 43.0 — 0 22.6 50 0 29.2 30 0 42.5 — 0 21.9  0 0 29.6  0 0KCCM11016P- 42.8 — 0 42.1  0 0 42.6  0 0 pcj7-xylAB(Er) 43.1 — 0 42.0  00 42.2  0 0 R.X: residual xylose (concentration of residual xylose afterreaction termination) (unit: g/l) R.G. residual glucose (concentrationof residual glucose after reaction termination)

As shown in Table 2, when the medium containing no xylose (glucose 100g/l) was used, concentration of L-lysine produced in KCCM11016P wasequivalent to that of KCCM11016P-pcj7-xylAB(Er). However, when themedium containing xylose (glucose 50 g/l+xylose 50 g/l, and glucose 70g/l+xylose 30 g/l) was used, the KCCM11016P strain produced L-lysine byconsuming only glucose whereas KCCM11016P-pcj7-xylAB(Er) producedL-lysine by consuming both glucose and xylose.

Example 6 Preparation of L-Lysine-Producing Strain Inserted with E.coli-Derived xylAB and Comparison of its Xylose-Utilizing Ability withthat of KCCM11016P-pcj7-xylAB(Ec) Strain

To compare the effects of improving xylose-utilizing ability betweenintroduction of the previously reported E. coli-derived xylAB andintroduction of Erwinia carotovora-derived XylAB of the presentinvention, a strain was prepared by introducing xylAB(Ec) intoKCCM11016P, and its xylose-utilizing ability and L-lysine productioncharacteristics were compared with those of the preparedKCCM11016P-pcj7-xylAB(Er).

To prepare a xylAB(Ec)-introduced strain, the pDZTn-pcj7-xylAB(Ec)recombinant vector prepared in Example 4 was transformed intoKCCM11016P, and through second crossover, a KCCM11016P-pcj7-xylAB(Ec)strain having xylAB(Ec) operably linked to pcj7 promoter inside thetransposon on the chromosome was obtained.

In the same manner as in example 3, to compare the xylose-utilizingability between KCCM11016P-pcj7-xylAB(Er) and KCCM11016P-pcj7-xylAB(Ec),they were cultured in the production medium containing glucose 50g/l+xylose 50 g/l as a carbon source, and their L-lysine productioncharacteristics were compared, and to examine the xylose-utilizingability, concentration of residual xylose in the culture broth wasmeasured at 15 hours (Table 3).

TABLE 3 R.X (g/L) Lysine 45 h 72 h 72 h KCCM11016P-pcj7-xylAB(Er) 8.0 042.8 7.2 0 42.2 KCCM11016P-pcj7-xylAB(Ec) 11.2 0 42.7 12.1 0 42.4 R.X:residual xylose (concentration of residual xylose after reactiontermination) (unit: g/l)

As shown in Table 3, the two strains showed equivalent L-lysineproductivity. The xylose consumption rate of KCCM11016P-pcj7-xylAB(Er)was faster than that of KCCM11016P-pcj7-xylAB(Ec), indicatingimprovement in fermentation productivity. That is, this result indicatesthat introduction of Erwinia carotovora-derived xylAB (Er) of thepresent invention shows more excellent effect of improving L-lysinefermentation productivity than introduction of the previous E.coli-derived xylAB(Ec).

Example 7 Development of KCCM10770P-Derived Strain Inserted with Erwiniacarotovora-Derived xylAB and Examination of Xylose-Utilizing Ability

To examine whether xylAB(Er) introduction exhibits the same effect inother L-lysine-producing strains than KCCM11016P, pDZTn-pcj7-xylAB(Er)was introduced into an L-lysine-producing strain KCCM 10770P (KoreanPatent No. 10-0924065) which was deposited to an InternationalDepositary Authority under the Budapest Treaty. After introduction by anelectric pulse method, a strain having xylAB(Er) with substitution ofone copy of pcj7 promoter inside the transposon on the chromosome wasobtained through second crossover, and the strain was named asCorynebacterium glutamicum KCCM10770P-pcj7-xylAB(Er).

Xylose-utilizing ability and L-lysine productivity of Corynebacteriumglutamicum KCCM10770P and Corynebacterium glutamicumKCCM10770P-pcj7-xylAB(Er) of the present invention were measured in thesame manner as in Example 3 (Table 4).

TABLE 4 Strain R.G R.X L-lysine KCCM10770P 0 50 23.8 0 50 24.4KCCM10770P-pcj7-xylAB(Er) 0 0 47.6 0 0 47.5 R.X: residual xylose(concentration of residual xylose after reaction termination) (unit:g/l) R.G: residual glucose (concentration of residual glucose afterreaction termination)

As shown in Table 4, when the L-lysine-producing strain KCCM10770P wasintroduced with xylAB(Er), it completely consumed xylose, unlike theparent strain that cannot utilize xylose, and its L-lysine productivitywas also increased.

This result supports that when Erwinia carotovora-derived xylAB isintroduced into various Corynebacterium sp. microorganisms as well asCorynebacterium sp. microorganism specified by a certain AccessionNumber, they completely consume xylose as a carbon source, therebyefficiently producing amino acids such as L-lysine.

Example 8 Development of KFCC10750-Derived Strain Inserted with Erwiniacarotovora-Derived xylAB and Examination of Xylose-Utilizing Ability

To examine whether xylAB(Er) introduction exhibits the same effect inother L-lysine-producing strains than KCCM11016P, pDZTn-pcj7-xylAB(Er)was introduced into an L-lysine-producing strain KFCC10750 (KoreanPatent No. 10-0073610). After introduction by an electric pulse method,a strain having xylAB(Er) with substitution of one copy of pcj7 promoterinside the transposon on the chromosome was obtained through secondcrossover, and the strain was named as Corynebacterium glutamicumKFCC10750-pcj7-xylAB(Er).

Xylose-utilizing ability and L-lysine productivity of Corynebacteriumglutamicum KFCC10750 and Corynebacterium glutamicumKFCC10750-pcj7-xylAB(Er) of the present invention were measured in thesame manner as in Example 3 (Table 5).

TABLE 5 Strain R.G R.X L-lysine KFCC10750 0 50 19.7 0 50 18.8KFCC10750-pcj7-xylAB(Er) 0 0 38.3 0 0 38.6 R.X: residual xylose(concentration of residual xylose after reaction termination) (unit:g/l) R.G: residual glucose (concentration of residual glucose afterreaction termination)

As shown in Table 5, when the L-lysine-producing strain KFCC10750 wasintroduced with xylAB(Er), it completely consumed xylose, unlike theparent strain that cannot utilize xylose, and its L-lysine productivitywas also increased.

This result supports that when Erwinia carotovora-derived xylAB isintroduced into various Corynebacterium sp. microorganisms as well asCorynebacterium sp. microorganism specified by a certain AccessionNumber, they completely consume xylose as a carbon source, therebyefficiently producing amino acids such as L-lysine.

Example 9 Development of CJ3P-Derived Strain Inserted with Erwiniacarotovora-Derived xylAB and Examination of Xylose-Utilizing Ability

To examine whether xylAB(Er) introduction exhibits the same effect inother L-lysine-producing strains than KCCM11016P, pDZTn-pcj7-xylAB(Er)was introduced into an L-lysine-producing strain CJ3P. CJ3P strain is aCorynebacterium glutamicum strain which has an ability to produceL-lysine by introduction of P458S, V59A, and T311I mutations into 3types of genes, pyc, horn, and lysC of the wild-type strain, based onthe technique reported by Binder et al. (Genome Biology 2102, 13:R40).After introduction by an electric pulse method, a strain havingxylAB(Er) with substitution of one copy of pcj7 promoter inside thetransposon on the chromosome was obtained through second crossover, andthe strain was named as Corynebacterium glutamicum CJ3P-pcj7-xylAB(Er).

Xylose-utilizing ability and L-lysine productivity of Corynebacteriumglutamicum CJ3P and Corynebacterium glutamicum CJ3P-pcj7-xylAB(Er) ofthe present invention were measured in the same manner as in Example 3(Table 6).

TABLE 6 Strain R.G R.X L-lysine CJ3P 0 50 4.0 0 50 4.5CJ3P-pcj7-xylAB(Er) 0 0 8.5 0 0 9.0 R.X: residual xylose (concentrationof residual xylose after reaction termination) (unit: g/l) R.G: residualglucose (concentration of residual glucose after reaction termination)

As shown in Table 6, when the L-lysine-producing strain CJ3P wasintroduced with xylAB(Er), it completely consumed xylose, unlike theparent strain that cannot utilize xylose, and its L-lysine productivitywas also increased.

This result supports that when Erwinia carotovora-derived xylAB isintroduced into various Corynebacterium sp. microorganisms as well asCorynebacterium sp. microorganism specified by a certain AccessionNumber, they completely consume xylose as a carbon source, therebyefficiently producing amino acids such as L-lysine.

Therefore, Corynebacterium sp. microorganism expressing xylAB(Er) isable to grow by utilizing xylose in a medium, and also able to produceL-lysine by utilizing xylose and glucose in a medium.

EFFECT OF THE INVENTION

When Corynebacterium sp. microorganism of the present invention able toproduce L-lysine by utilizing xylose is used, L-lysine can be producedby using xylose as the second most abundant lignocellulosic carbohydratein nature. Therefore, the microorganism can be widely used for theefficient and economical production of L-lysine.

1. A modified Corynebacterium sp. microorganism able to produce L-lysineby utilizing xylose, wherein activities of Erwinia carotovora-derivedxylose isomerase (XylA) and xylulokinase (XylB) are introduced.
 2. Themicroorganism according to claim 1, wherein XylA comprises an amino acidsequence of SEQ ID NO: 1, and XylB comprises an amino acid sequence ofSEQ ID NO:
 2. 3. The microorganism according to claim 1, wherein XylA isencoded by a polynucleotide sequence of SEQ ID NO: 3, and XylB isencoded by a polynucleotide sequence of SEQ ID NO:
 4. 4. Themicroorganism according to claim 1, wherein the microorganism isCorynebacterium glutamicum.
 5. The microorganism according to claim 1,wherein the introduction of XylA and XylB activities is carried out byinserting a polynucleotide including nucleotide sequences encoding XylAand XylB into a chromosome, introducing a vector system including thepolynucleotide into the microorganism, introducing a potent promoter toupstream of the nucleotide sequences encoding XylA and XylB, introducingXylA and XylB with a modified promoter, or introducing the modifiednucleotide sequences encoding XylA and XylB.
 6. A method for producingL-lysine, comprising the steps of: (i) culturing the modifiedCorynebacterium sp. microorganism able to produce L-lysine by utilizingxylose according to claim 1 in a culture medium containing xylose as acarbon source so as to obtain a culture broth; and (ii) recoveringL-lysine from the culture broth.